Cell death and autophagy in prion diseases (transmissible spongiform encephalopathies)

Neuronal autophagy, like apoptosis, is one of the mechanisms of programmed cell death. In this review, we summarize current information about autophagy in naturally occurring and experimentally induced scrapie, Creutzfeldt-Jakob disease and Gerstmann-Str?ussler-Scheinker syndrome against the broad background of neural degenerations in transmissible spongiform encephalopathies (TSEs). Typically a sequence of events is observed: from a part of the neuronal cytoplasm sequestrated by concentric arrays of double membranes (phagophores); through the enclosure of the cytoplasm and membrane proliferation; to a final transformation of the large area of the cytoplasm into a collection of autophagic vacuoles of different sizes. These autophagic vacuoles form not only in neuronal perikarya but also in neurites and synapses. On the basis of ultrastructural studies, we suggest that autophagy may play a major role in transmissible spongiform encephalopathies and may even participate in the formation of spongiform change.     

Astrocytosis and amyloid deposition in scrapie-infected hamsters

In scrapie infection, prion protein (PrP^Sc) is localized in areas where there is neurodegeneration and astrocytosis. It is thought that PrP^Sc is toxic to neurons and trophic for astrocytes. In our study, paraffin sections from scrapie infected (263K and 139H) and control hamsters were examined with histological and immunocytochemical staining. We found that PrP^Sc was present in the ependymal cells of both 263K- and 139H-infected hamsters. In 139H-infected hamsters, PrP^Sc was found in the cytoplasm of neurons in cerebral cortex and in hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. In contrast, neuronal cytoplasm and nuclei, were positive for PrP^Sc in most areas such as cortex, hippocampus, and thalamus in 263K-infected hamsters. Many aggregations of PrP^Sc could be seen in the cortex, hippocampus, substantia nigra and around the Pia mater, corpus callosum, fimbria, ventricles, and blood vessels in sections from 139H- and/or 263K-positive animals. Furthermore, PrP^Sc was also co-localized with glial fibrillary acidic protein (GFAP) in many reactive astrocytes (approximately 90%) in certain areas such as the hippocampus in 263K-infected hamsters, but not 139H-infected hamsters. The patterns of astrocytosis and PrP^Sc formation were different between 139H- and 263K-infected hamsters, which may be used for a diagnosis purpose. Our results suggest a hypothesis that multiple cell-types are capable of PrP^Sc production. Our results also confirm that reactive astrocytes can produce and/or accumulate PrP^Sc during some scrapie strain infections. The findings suggest a `snowball effect', that is: astrocytosis might play an important role in amyloidosis, while amyloidosis may induce further astrocytosis at least in 263K-infected hamsters.     

Differential expression of Bax and Bcl-2 in the brains of hamsters infected with 263K scrapie agent

To study the mechanism(s) of neuronal cell death during scrapie infection, we investigated the expression of Bax and Bcl-2 in brains of hamsters infected with 263K scrapie agent. The expression of Bcl-2 mRNA was significantly decreased in the brains of 263K scrapie-infected hamsters compared with controls, whereas the expression levels of Bax mRNA were significantly increased in scrapie-infected brain. The levels of Bax and Bcl-2 proteins in brains of scrapie and control animals reflected the difference in mRNA levels. Immunoreactivity for Bax and Bcl-2 were found predominantly within neurons. In scrapie-infected brains, the number of neuronal cells positive for Bcl-2 was significantly lower in the hippocampal CA3 region and was decreased in the cerebral cortex, whereas the number of neuronal cells positive for Bax was significantly increased in both regions. The possibility that differential regulation of Bax and Bcl-2 expression may play an important role in neuronal cell death induced by scrapie infection is discussed.     

Altered plasma membranes in experimental scrapie

Summary The status spongiosus in the cerebral cortex of mice affected with two different strains of scrapie virus corresponded to focally swollen perikaryal cytoplasm of nerve cells and astrocytes, to swollen neuronal and astrocytic processes and to membrane-bounded vacuoles within pre- and postsynaptic neuronal terminals. The swollen cytoplasm contained uniformly dispersed, finely granulo-filamentous material. A few enlarged dendrites were filled with fragments of membranes or 350 Å wide vesicular and tubular structures suggestive of virus particles. Ruptured plasma membranes and curled fragments of membranes were seen around cleared cytoplasmic regions and within membrane-bounded vacuoles. Neurons or astrocytes that lined affected cells or processes frequently showed similar changes. Confluence of swollen cells or processes occurred after dissolution of their adjacent plasma membranes. Astrocytes reacted to the injury by proliferation whereas nerve cells degenerated. The findings are compared to those seen in other subacute spongiform virus encephalopathies, i. e., mink encephalopathy, Kuru and Creutzfeldt-Jakob disease. The characteristic vacuolar degeneration of nerve cells in these diseases which is associated with fragmentation and accumulation of plasma membranes is discussed with reference to the peculiar properties of the scrapie virus.This investigation was supported in part by United States Public Health Research Grant NS-09053 from the National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland.     

Scrapie as a model for neuroaxonal dystrophy: ultrastructural studies

Neuritic degeneration is a prominent ultrastructural feature of scrapie in hamsters. To investigate the morphogenesis of neuritic degeneration, we examined brain tissues from hamsters infected with the 263K strain of scrapie virus and from age-matched controls at varying intervals following intracerebral inoculation. Dystrophic neurites--defined as dendrites, axonal preterminals, and myelinated axons containing mitochondria and pleomorphic, electron-dense inclusion bodies--were found as early as 2 weeks postinoculation. Their numbers increased with the incubation period, and their highest density was observed at the terminal stage of disease. Occasionally, small clusters of these structures formed neuritic plaques. Such dystrophic neurites were only rarely seen in brains of uninfected hamsters. Experimental scrapie thus provides an animal model for human neuroaxonal dystrophies. In addition, since this model allows predictable formation of brain amyloid, it may serve as a model for the study of neuronal aging and Alzheimer's disease.     

Increase of acidic fibroblast growth factor in the brains of hamsters infected with either 263K or 139H strains of scrapie

Scrapie is the archetypal unconventional slow infection disease. It has been shown that hamsters injected intracerebrally with scrapie strains 139H or 263K show extensive astrocytosis and that the induced reactive astrocytes produce a variety of factors that can affect brain function. Acidic fibroblast growth factor (aFGF) belongs to a family of growth factors that show a high affinity for heparin sulfate proteoglycans. In the current study, we have used immunohistochemistry to investigate the distribution of aFGF in scrapie-infected brain; we observed a low level of aFGF immunoreactivity (ir-aFGF) in ependymal cells and in a few neurons in the hypothalamus of control hamsters. In contrast, in scrapie-infected hamsters, there was an increase of ir-aFGF in a number of cell types, including neurons, pericytes, astrocytes, and ependymal cells. In 139H-infected hamsters, ir-aFGF staining in astrocytes, neurons and neuropil areas of the cortex, hippocampus, thalamus, and hypothalamus was greater than the staining in control animals. For 263K animals, astrocytic ir-aFGF staining was significantly greater than in either control or 139H-infected hamsters in the following regions: cortex, putamen, corpus callosum, thalamus, hypothalamus, fimbria, hippocampus, subependymal areas, and amygdala. In addition, there was a significant increase in neuronal ir-aFGF in the CA1 hippocampal area and in the amygdala. Our results suggest that neurons and astrocytes can produce and/or absorb aFGF during scrapie infection. These findings indicate that aFGF might play an important role in neuronal protection and in astrocytosis in scrapie-infected hamsters.     

Neuronal autophagic vacuoles in experimental scrapie and Creutzfeldt-Jakob disease

Summary We report the presence of autophagic vacuoles (AV) in neuronal perikarya and neuronal processes of rodents with experimental scrapie and Creutzfeldt-Jakob disease. AV were composed of sequestrated cytoplasmic areas containing ribosomes and occasionally mitochondria and small secondary vacuoles. The formation of AV may contribute to neuronal degeneration and ultimately to neuronal loss.P. P. Liberski is a recipient of a fellowship from the Fogarty International Center and a grant from the Polish Academy of Sciences (VIII/40)     

Scrapie inoculation of mice: light and electron microscopy of the superior colliculi

Summary Ultrastructural examination of the superior colliculi of mice intraocularly inoculated with the ME7 strain of scrapie showed vacuolation early in the course of infection. Brains were examined between 85–260 days after monocular inoculation with scrapie. The mean incubation period for the development of clinical disease was 302 days. Vacuolation was seen initially in the contralateral superior colliculus and subsequently in the ipsilateral colliculus. In coded trails light microscopical vacuolation was seen from 218 days but ultrastructural examination showed that sparse vacuoles were inconsistently present in either or both of the ipsilateral and contralateral colliculi from 85 days; frequent vacuoles were seen from 190 days. Scrapie-induced vacuoles were differentiated from vacuoles present in control tissue by the presence of loculation or by a limiting double membrane which showed protrusion or proliferation of the innermost lamella. Vacuolation was seen in neuronal perikarya, myelinated fibres, dendrites and axonal presynaptic terminals. Vacuoles of myelinated fibres were observed within myelin and possibly also in the inner tongue of oligodendroglial cytoplasm. Whorled membrane configurations were also seen. Tubulovesicular particles, 40 nm in diameter, were recognised in two scrapie-infected mice. It is suggested that some scrapie vacuoles arise as a result of incorporation of abnormal membrane into organelles, possibly mitochondria, in neuronal perikarya and neurites and probably also within oligodendroglial cytoplasm and myelin.     

Appearance of tubulovesicular structures in experimental Creutzfeldt-Jakob disease and scrapie preceeds the onset of clinical disease

Summary We have consistently observed tubulovesicular structures in brain tissues during the terminal stages of naturally occurring and experimentally induced spongiform encephalopathies, irrespective of the host species and virus strain. In NIH Swiss mice inoculated intracerebrally or intraocularly with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) virus, tubulovesicular structures, measuring 20–50 nm in diameter, were particularly prominent in dilated, pre-and postsynaptic neuronal processes, occasionally being mixed with synaptic vesicles. These structures appeared 13 weeks following intracerebral inoculation, 5 weeks before the onset of clinical signs, when spongiform changes were also detected. The number and density of tubulovesicular structures increased steadily during the course of clinical disease, and were particularly abundant in mice 47 to 51 weeks after intraocular inoculation. In hamsters infected with the 263 K strain of scrapie virus, these structures were initially detected 3 weeks following intracerebral inoculation and increased dramatically at 10 weeks postinoculation. The appearance of tubulovesicular structures before the onset of overt disease in mice inoculated with CJD virus by either the intracerebral or intraocular route, and before the appearance of other neuropathological changes in hamsters infected with scrapie virus, indicate that they represent either a part or aggregate of the infectious virus or a pathological product of the infection.Presented in part at the 64th annual meeting of the American Association of Neuropathologists, held in Charleston, South Carolina, June 9–12, 1988 and at the 7th annual meeting of the American Society for Virology, Austin, Texas, June 12–16, 1988. Dr. Pawel P. Liberski is a recipient of a fellowship from the Fogarty International Center and a grant from the Ministry of Health and Social Welfare, Poland     

Ultrastructural findings in pigs experimentally infected with bovine spongiform encephalopathy agent

We report here an electron microscopic study of selected nervous system tissues from pigs infected experimentally with the agent of bovine spongiform encephalopathy (BSE). Generally, the ultrastructural neuropathology of BSE-affected pig brain resembled that of BSE-affected cattle brain. Spongiform change, in the form of membrane-bound vacuoles separated by septae into secondary chambers, dominated the pathology. Numerous astrocytic processes were visible in close conjunction with elongated microglial cells. Neuronal degeneration presented as either dystrophic neurites or by the formation of autophagic vacuoles. Altered subcellular organelles: mitochondria, electron-dense bodies, autophagic vacuoles, neurofilaments and "branching-cisterns" accumulated in abnormal neurites. Autophagic vacuoles appeared as neuronal cytoplasm of increased electron-density sequestrated by intracytoplasmic membranes. Tubulovesicular structures were numerous, particularly in the cerebellum. Unusual crystalloids were observed in the white matter. In conclusion, experimental BSE in pigs demonstrated ultrastructural pathology in keeping with that observed in other spongiform encephalopathies.     

An electron microscopic study of inclusion bodies in synaptic terminals of scrapie-infected animals

Summary Inclusion bodies consisting of vesicles of about 25 nm diameter and occurring in the synaptic terminals of scrapie-infected animals have been described by a number of people. In the present study these inclusion bodies were looked for in the neocortex, hippocampus and corpus callosum in a variety of strains of mice (C3H, LM, RIII, IM, VL) infected with different strains of scrapie agent (22C, 79A, ME7, 87V) after intracerebral inoculation. In plaque-bearing models of scrapie, terminals containing synaptic inclusion bodies were frequently found surrounding the amyloid plaque cores in the neocortex but not in the corpus callosum. In non-plaque-bearing models, terminals containing synaptic inclusion bodies were found in the neuropil of the neocortex and hippocampus. For all models, these bodies were either presynaptic or postsynaptic but were not, as a rule, found on both sides of the same synapse. Fibrillary material was frequently seen in the postsynaptic terminals containing the inclusion bodies in both the plaque- and non-plaque-bearing models. On one occasion fibrillary material was seen, together with the inclusion bodies, in a neuron cell body. Inclusion bodies were also seen in the neocortex of hamsters infected with the 263K strain of scrapie agent and a Cheviot sheep infected with the ME7 strain of agent. The inclusion bodies and the fibrillary material were thought to be derived from the breakdown of neurotubules.     

Astrocytosis and proliferating cell nuclear antigen expression in brains of scrapie-infected hamsters

Scrapie is a neurodegenerative disease in sheep and goats. Neuropathological examination shows astrocytosis. One issue is whether the astrocytosis seen in scrapie is a function of an increase in reactivity of individual cells, or whether there is actual replication of astrocytes. We used double-label immunohistochemistry for proliferating cell nuclear antigen (PCNA) and for glial fibrillary acidic protein (GFAP) to determine the mitotic state of cells and to confirm their identity as astrocytes. Brain sections from hamsters (strain LVG/LAK) infected with 139H or 263K scrapie isolates were examined. GFAP immunostaining was increased in astrocytes in most regions of the brains of scrapie-infected hamsters. These qualitative observations were confirmed by computerized image analysis quantification. A proportion of the hypertrophic astrocytes (0.5–10.8%, depending on specific location) were PCNA immunoreactive. The PCNA-immunopositive astrocytes were most frequently found in cerebral cortex, corpus callosum, subependymal areas, fimbria, caudate, thalamus, hypothalamus, hippocampus, and dentate gyrus. Our results suggest that the astrocytosis seen in scrapie-infected animals is, at least in part, owing to actual replication of astrocytes in these animals. We hypothesize that the astrocytes may be an important locus for the disease process.     

TRANSMISSIBLE MINK ENCEPHALOPATHY (TME) IN CHINESE HAMSTERS: IDENTIFICATION OF TWO STRAINS OF TME AND COMPARISONS WITH SCRAPIE

TME from a single source was transmitted by intracerebral injection to Chinese hamsters, producing clinical disease in all seven animals after incubation periods of over 600 days. The brain from each of the primary cases was used to establish separate intracerebral passage-lines of TME and this led to the isolation of two different strains of agent, designated 333K and 333W. These strains were easily distinguished by the incubation periods they produced (about 130 and 230 days, respectively) under standard conditions of infection, and by the characteristic profiles of vacuolation seen in different regions of the brain. Comparisons were made with a strain of scrapie passaged in Chinese hamsters, designated 34W, which could be distinguished from both strains of TME. Nevertheless the properties of the scrapie and TME strains overlapped, with one of the TME strains (333K) resembling the 34W strain of scrapie in Chinese hamsters more closely than the other TME strain (333W). These similarities strengthen the view that TME and scrapie are caused by a similar type of infectious agent. The very large 'species barrier effect' on transmitting TME to Chinese hamsters was in marked contrast to the minimal effect seen with scrapie and an explanation for this is suggested. Two interesting pathological features of the study were (a) the severe loss of pyramidal cells produced in the hippocampus by the 34W strain of scrapie, and (b) the focal, symmetrical vacuolation of the thalamus caused by 333K TME.     

Photoreceptor degeneration during infection with various strains of the scrapie agent in hamsters

Hamsters were inoculated intracerebrally with the 22C, 79A, and ME7 strains of the scrapie agent to compare the effects on the retina with those caused by strain 263K. The animals developed clinical signs of encephalopathy. Photoreceptor degeneration occurred in all experimental animals. The changes were similar to those seen in animals infected with the 263K strain of scrapie although somewhat more variable and less extensive.     

Neuroinvasion of the 263K scrapie strain after intranasal administration occurs through olfactory-unrelated pathways

The olfactory system has been implicated in the pathogenesis of transmissible spongiform encephalopathies (TSEs). To examine this issue and identify the pattern of TSE agent spread after intranasal administration, we inoculated a high-infectious dose of neurotropic scrapie strain 263K into the nasal cavity of Syrian hamsters. All animals allowed to survive became symptomatic with a mean incubation period of 162.4 days. Analysis at different time points revealed deposition of the pathological prion protein (PrP^TSE) in nasal-associated lymphoid tissues in the absence of brain involvement from 80 days post-infection (50% of the incubation period). Olfactory-related structures and brainstem nuclei were involved from 100 days post-inoculation (62% of the incubation period) when animals were still asymptomatic. Intriguingly, vagal or trigeminal nuclei were identified as early sites of PrP^TSE deposition in some pre-symptomatic animals. These findings indicate that the 263K scrapie agent is unable to effectively spread from the olfactory neuroepithelium to the olfactory-related structures and that, after intranasal inoculation, neuroinvasion occurs through olfactory-unrelated pathways.     

Scrapie-specific neuronal lesions are independent of neuronal PrP expression

In the transmissible spongiform encephalopathies (TSE), accumulation of the abnormal disease-specific prion protein is associated with neurodegeneration. Previous data suggested that abnormal prion protein (PrP) could induce neuronal pathology only when neurons expressed the normal form of PrP, but conflicting evidence also has been reported. Understanding whether neuronal PrP expression is required for TSE neuropathological damage in vivo is essential for determining the mechanism of TSE pathogenesis. Therefore, these experiments were designed to study scrapie pathogenesis in vivo in the absence of neuronal PrP expression. Hamster scrapie (strain 263K) was used to infect transgenic mice expressing hamster PrP in the brain only in astrocytes. These mice previously were shown to develop clinical scrapie, but it was unclear whether the brain pathology was caused by damage to astrocytes, neurons, or other cell types. In this electron microscopic study, neurons demonstrated TSE-specific pathology despite lacking PrP expression. Abnormal PrP was identified around astrocytes, primarily in the extracellular spaces of the neuropil, but astrocytes showed only reactive changes and no damage. Therefore, in this model the pathogenesis of the disease appeared to involve neuronal damage associated with extracellular astrocytic accumulation of abnormal PrP acting upon nearby PrP-negative neurons or triggering the release of non-PrP neurotoxic factors from astrocytes.     

Neuronal autophagy in experimental scrapie

Summary In this study we report the formation of giant autophagic vacuoles (AV) in neurons in experimental scrapie in hamsters. Autophagy is an important step in the cellular turnover of proteins and organelles. It is known to occur in neurons under physiological as under pathological conditions. Giant AV, however, are seen very rarely only in pathological states. In our model AV are much more numerous after intracerebral (i.c.) transmission of the scrapie agent than after the transmission via the intraperitoneal route which points to a correlation between the intensity of the process and the period of incubation. As the appearance of the AV in our model is correlated chronologically with that of scrapie-associated fibrils, at least after i.c. transmission, the process may be related to a disturbance of cellular protein metabolism and, thus, to the processing of prion protein.     

Muscle fiber degradation in distal myopathy with rimmed vacuoles

Late-onset distal myopathy showed numerous rimmed vacuoles with the same properties as autophagic vacuoles. Electron microscopy showed numerous degenerated mitochondria, glycogen, or cell membranes in rimmed vacuoles, but no evidence that these vacuoles engulfed and contained intact or partially disrupted myofibrils. Immunostaining for myosin, -actinin, and actin, however, was sometimes positive within the vacuoles. Compared to the control muscle, there was increased staining activity by calpain around the rimmed vacuoles or in the cytoplasm of mainly atrophic fibers. The result seems to indicate an increase of calpain activity in these muscle fibers. We hypothesize that the myofibrils as well as mitochondria, glycogen, or cell membranes in this myopathy are degraded finally through a lysosomal autophagic process. However, the breakdown of the myofibrils may be not initiated by lysosomal activation; rather it may be the result of extralysosomal processes such as the calpain system.Supported by Research Grant 2A-1-10 for Nervous and Mental Disorders from the Ministry of Health and Welfare and by a Grant-in-Aid (04670493) for Scientific Research from the Ministry of Education, Science and Culture of Japan     

Effects of the polyene antibiotic derivative MS-8209 on the astrocyte lysosomal system of scrapie-infected hamsters

Amphotericine B (AmB), a macrolide polyene antibiotic, is one of a few drugs that has shown therapeutic properties in scrapie-infected hamster. Its beneficial effect on survival time is mostly marked when animals are treated with its derivative MS-8209. To explore the MS-8209 effect at the cellular level, we investigated at the light and electron microscopy levels, the sequential appearance and distribution of PrP concurrently with histopathological changes in hamsters that were infected intracerebrally with the 263 K scrapie strain and treated or not with the drug. The first histopathological modifications and PrP immunostaining were observed in the thalamus and at the inoculation site where the drug caused a delay in the appearance of lesions and PrP accumulation. Using immunoelectron microscopy, at 70 d postinfection, the inoculation site of untreated animals showed an accumulation of PrP in plaque areas constitued by filaments mixed with alterated membrane structures and in developed lysosomal system of reactive astrocytes. Most of the numerous lysosomes containing PrP showed intra-organelle filaments. In contrast, in MS-8209 treated animals, the number of lysosomes was significantly lower (p<0.0038), with very few organelles harboring PrP. Our results suggest that in this scrapie model, MS-8209 treatment delays the disease by preventing the replication of the scrapie agent at the inoculation site where the astrocytes appear to be the first cells producing abnormal PrP. The lysosomal system of these astrocytes could constitute a privileged target for MS-8209.     

Optic nerve degeneration in hamsters experimentally infected with scrapie

Optic nerve degeneration occurred late in hamsters experimentally infected with scrapie. Disorganization and degeneration of myelin lamellae occurred in many of the axons of the orbital and prechiasmal regions of the nerve; a few axons showed swelling and degeneration of cytosol. Intracellular vacuoles similar to those seen in the brain were observed only in the nonmyelinated intraocular portion of the optic nerve.